The deposition in the brain parenchyma of the small peptide ABeta as insoluble Beta-amyloid plaque is an invariant feature of Alzheimer's disease (AD). Although genetic defects in the rare early-onset familial forms of the disease have been shown to increase ABeta production, little is known about the events that cause amyloid deposition in the much more common, sporadic form of AD. We have recently shown that calpains, cytosolic, Ca++-activated neutral proteases, regulate ABeta formation from the amyloid precursor protein (APP) by altering the intracellular trafficking of this protein. In Aim 1 of this proposal, we will examine neuron-like cells in culture to test our hypothesis that calpain activity modulates the trans-Golgi network/plasma membrane/early endosome distribution of APP via its regulation of the endocytic recycling pathway. We will determine further whether this is the mechanism by which calpain activity affects APP processing, Beta-cleavage of APP and ABeta generation. Additionally, we will extend our observation that early endosomes contain many Beta-cleaved C-terminal fragments of APP (BetaCTF), the rate limiting substrate for ABeta generation from APP, and determine the trafficking of BetaCTFs between various intracellular compartments. In Aim 2, we will confirm the importance of the early endosome for BetaCTF generation. These studies have important clinical implications, in part because the protease responsible for BetaCTF generation (BACE) is an attractive target for AD therapeutics, and understanding its site(s) of action on APP may be key in developing therapeutically useful inhibitors. In Aim 3, we will extend these in vitro studies into the intact central nervous system and further exploit our recent discovery that ABeta42 production - a particularly pathogenic species of ABeta - is dramatically increased following calpain inhibition to initiate Beta-amyloid plaque deposition in vivo. Our proposed studies will expand upon existing mouse models of AD pathology, will directly link calpain activity - which has been shown to be altered in AD - to in vivo alterations in APP trafficking and ABeta production, and will determine whether calpain inhibition can initiate Beta-amyloid plaque formation. Since calpain inhibitors are being pursued for the treatment of CNS ischemia such as stroke, the clinical implications of such a finding are significant: inhibiting calpain activity may have currently unappreciated deleterious consequences.